This proposal s long term goal is to determine the molecular mechanisms governing early mammalian eye formation. A key gene involved in oculogenesis is Pax6: human and mouse Pax6 mutations result in aniridia and Small eye, while mutations in a Drosophila Pax6 homolog result in the eyeless phenotype. Strikingly, Pax6 misexpression in Drosophila produces ectopic eyes, implying that vertebrate and invertebrate eye formation utilize similar, evolutionarily conserved Pax6-dependent genetic hierarchies. During the last grant period, we defined the Pax6 gene product as a transcriptional activator, elucidated the DNA binding properties of its paired domain and identified the Msx and Eya gene families as important downstream components of the eye forming regulatory hierarchy. We also identified two murine Pax6 promoters and several Pax6 regulatory elements which direct expression in different parts of the developing eye. In addition, we have found that Drosophila eyeless and mouse Pax6 regulatory elements appear to function to some extent interchangeably in transgenic mice and flies, suggesting that the upstream regulation of Pax6 is also evolutionarily conserved. In this renewal application, we seek to further elucidate the Pax6- dependent genetic pathway(s) controlling early mammalian eye development. In the first specific aim, we will go upstream by further analyzing regulatory elements controlling Pax6 ocular expression and by identifying the transcription factors that interact with them. In the second specific aim, we will focus on Pax6 downstream targets and clarify the relationship between them in the developing lens placode. In the third aim, we will determine if Msx and Eya are directly regulated by Pax6 and clarify the details of a strong protein-protein interaction we have detected between the highly conserved Eya domain and the Msx and Six homeoproteins. Finally, in the fourth aim, we will use try to complement Pax6 function in prospective lens ectoderm of Pax6-deficient Sey/Sey mice with various Pax6 transgenes and downstream target genes, thus reconstituting early steps of lens induction. This latter goal, although ambitious, has already been accomplished by others in a limited, empiric fashion in insects, fish and amphibians. The proposed work will identify factors controlling Pax6 ocular expression and downstream Pax6 regulatory targets, clarify key details of the emerging Pax6- hierarchy at the molecular level and test this regulatory hierarchy in vivo.